KDO aldolase and condensation reactions employed therewith

ABSTRACT

Aureobacterium barkerei  strain KDO-37-2 (ATCC 49977) and KDO aldolase (EC 4.1.2.23) isolated therefrom are disclosed. The KDO aldolase is further disclosed to have a broad substrate specificity with respect to its reverse reaction, i.e. the condensation of aldoses with pyruvate to form a wide range of 2-keto-3-deoxy-onic acids, including 2-keto-3-deoxy-nonulosonic acid, 2-keto-3-deoxy-octulosonic acid, 2-keto-3-deoxy-heptulosonic acid, and 2-keto-3-deoxy-hexulosonic acid. In particular, 3-deoxy-D-manno-2-octulosonic acid (D-KDO), a vital component of lipopolysaccharides found in the bacterial outer membrane may be synthesized from D-arabinose and pyruvate in 67% yield. Additionally, protected forms of the KDO aldolase products, e.g. hexaacetyl 2-keto-3-deoxy-nonulosonic acid and pentaacetyl 2-keto-3-deoxy-octulosonic acid, may be decarboxylated to form the corresponding 2-deoxy-aldoses, e.g. 2-deoxy-octulose and 2-deoxy-heptulose respectively.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional application of application Ser. No. 08/767,182,filed Dec. 16, 1996, issued Feb. 9, 1999 as U.S. Pat. No. 5,869,316,which is a divisional application of application Ser. No. 08/328,739,filed Oct. 25, 1994, issued Dec. 17, 1996 as U.S. Pat. No. 5,585,261,which is a divisional application of application Ser. No. 07/993,140,filed Dec. 18, 1992, issued Oct. 25, 1994 as U.S. Pat. No. 5,358,859.

GOVERNMENT RIGHTS

This invention was made with government support under Grant No. GM 44154awarded by the National Institutes of Health. The U.S. government hascertain rights in the invention.

DESCRIPTION Technical Field

The invention relates to KDO aldolase (EC 4.1.2.23) having a broadsubstrate specificity with respect to its reverse reaction and tocondensation reactions employing such KDO aldolase for synthesizing abroad range of 6-9 carbon 2-keto-3-deoxy-onic acids, viz.2-keto-3-deoxy-hexulosonate, 2-keto-3-deoxy-heptulosonate,2-keto-3-deoxy-octulosonate, and 2-keto-3-deoxy-nonulosonate. Moreparticularly, the invention relates to Aureobacterium barkerei strainKDO-37-2 (ATCC 49977), to KDO aldolase produced by and isolated fromsuch bacteria, to the employment of such KDO aldose with respect to thesynthesis of 2-keto-3-deoxy-onic acids such as3-deoxy-D-manno-2-octulosonic acid (D-KDO) and to the use of protectedforms of such 2-keto-3-deoxy-onic acids for the production of 7 and 8carbon aldoses by means of radical mediated decarboxylation.

BACKGROUND OF THE INVENTION

2-Keto-3-deoxy-octulosonic acid (KDO) appears as a ketosidic componentof all Gram-negative bacteria for which a KDO determination has beenmade. More particularly, 3-deoxy-D-manno-2-octulosonic acid (D-KDO) iswidely found in Gram-negative bacteria. KDO is incorporated intolipopolysaccharides and is localized, as such, within the outer membranecompartment of Gram-negative bacteria. KDO appears to be a vitalcomponent of Gram-negative bacteria. KDO can also occur as an acidicexopolysaccharide. In such instances, the KDO can serve as part of aK-antigen.

As illustrated in FIG. 7, the biosynthetic incorporation of KDO intolipopolysaccharides consists of two steps, i.e.:

1. Activation of KDO to form CMP-KDO by means of CMP-KDO synthetase (EC2.7.7.38); and then

2. Coupling of the activated CMP-KDO to lipid A precursor to form lipidA-KDO by means of KDO transferase.

The rate-limiting step with respect to the biosynthesis of KDOcontaining lipopolysaccharides is the activation of the KDO moiety,i.e., the formation of CMP-KDO. Accordingly, inhibitors of CMP-KDOsynthetase are potentially useful as antibacterial agents.

Several chemical and enzymatic synthetic routes have been developed forthe synthesis of KDO and its analogs. One route for the chemicalsynthesis of KDO employs Cornforth's method. (Ghalambor, M. et al. J.Biol. Chem. 1966, 241, 3207 and Hershberger, C. et al. J. Biol. Chem.1968, 243, 1585.) The chemical synthesis of KDO produces multipleenantiomers. In order to obtain enantiomerically pure D-KDO, aseparation step must be incorporated into the chemical synthetic route.

Synthetic routes employing enzymes are more stereospecific than chemicalsynthetic routes. An enzymatic synthetic route employing KDO-8 phosphatesynthase and KDO-8-P phosphatase as catalysts and arabinose-5-P and PEPas substrates is illustrated in FIG. 7. (Bednarski, M. et al.Tetrahedron Letters 1988, 29, 427.) An alternative enzymatic syntheticroute employs sialic acid aldolase. (Augé, C. et al. Tetrahedron 1990,46, 201.)

An enzymatic synthetic route employing the reverse reaction of KDOaldolase for a micromolar scale synthesis of KDO is disclosed byGhalambor. (Ghalambor, M. et al., J. Biol. Chem. 1966, 241, 3222.) Thesynthetic route described by Ghalambor employs KDO aldolase isolatedfrom Aerobacter cloacae. The reverse reaction of KDO aldolase is drivenby employing high substrate levels, i.e. high concentrations ofD-arabinose and D-pyruvate. Ghalambor discloses that there is a 41%yield with this enzyme and narrow substrate specificity.

KDO aldolase (EC 4.1.2.23) is known to be inductively produced byseveral bacteria, viz. Escherichia coli, strains 0111, B, and K-12,Salmonella typhimurium, Salmonella aldelaide, and Aerobacter cloacae.Ghalambor discloses that all of these known KDO aldolases havecomparable activities. For example, all of these KDO aldolases hydrolyze3-deoxy-D-manno-2-octulosonic acid to form D-arabinose and pyruvate in aforward reaction. As indicated above, Ghalambor also discloses thatknown KDO aldolase may be employed in a reverse reaction to condenseD-arabinose and pyruvate to form 3-deoxy-D-manno-2-octulosonic acid. Thesubstrate specificity of known KDO aldolases with respect to thisreverse reaction is confined to D-arabinose and has been specificallyshown to lack a measurable specificity for D-ribose in connection withthis reverse reaction.

What was needed was an enzymic synthetic route for the production of awide range of 2-keto-3-deoxy-onic acids and analogs thereof potentiallyhaving activity as inhibitors of CMP-KDO synthetase.

What was also needed was a method of converting 2-keto-3-deoxy-onicacids to high-carbon 2-deoxy aldoses.

SUMMARY OF THE INVENTION

Aureobacterium barkerei strain KDO-37-2 (ATCC 49977) and KDO aldolase(EC 4.1.2.23) isolated therefrom are disclosed therein. KDO aldolasecatalyzed condensation employing this enzyme has been demonstrated to beeffective for the synthesis of KDO and analogs. The reactions arestereospecific with formation of a new R-stereocenter at C-3 fromD-arabinose and related substrates. Decarboxylation of the aldolaseproducts provides a new route to heptose and octose derivatives.

Unlike known KDO aldolases which have a narrow substrate specificity,the KDO aldolase isolated from this source is disclosed to have a verywide substrate specificity with respect to catalyzing its reversereaction, i.e. the condensation of aldoses with pyruvate. In particular,3-deoxy-D-manno-2-octulosonic acid (D-KDO) can be synthesized fromD-arabinose and pyruvate in 67% yield. Furthermore, studies with respectto the substrate specificity of the enzyme using more than 20 naturaland unnatural sugars indicate that this enzyme widely accepts trioses,tetroses, pentoses and hexoses as substrates, especially the ones with Rconfiguration at 3 position. The substituent on 2 position has littleeffect on the aldol reaction. Nine of these substrates are submitted tothe aldol reaction to prepare various 2-keto-3-deoxy-onic acids,including D-KDO, 3-deoxy-D-arabino-2-heptulosonic acid (D-DAH),2-keto-3-deoxy-L-gluconic acid (L-KDG), and3-deoxy-L-glycero-L-galacto-nonulosonic acid (L-KDN). The attack ofpyruvate appears to take place on the re face of the carbonyl group ofacceptor substrates, a facial selection complementary to sialic acidaldolase (si face attack) reactions. The aldolase products can beconverted to aldoses via radical-mediated decarboxylation. For example,decarboxylation of pentaacetyl KDO and hexaacetyl neuraminic acid givespenta-O-acetyl-2-deoxy-β,3-D-manno-heptose andpenta-O-acetyl-4-acetamido-2,4-dideoxy-β-D-glycero-D-gala cto-octose,respectively.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates saccharides having good specificity for KDO aldolaseisolated from Aureobacterium barkerei KDO-37-2.

FIG. 2 illustrates saccharides having fair specificity for KDO aldolaseisolated from Aureobacterium barkerei KDO-37-2.

FIG. 3 illustrates saccharides having poor specificity for KDO aldolaseisolated from Aureobacterium barkerei KDO-37-2.

FIG. 4 illustrates the stereochemistry of the aldol condensationcatalyzed by KDO aldolase isolated from Aureobacterium barkereiKDO-37-2.

FIG. 5 illustrates the ¹H NMR spectrum of 6, the product from2-deoxy-D-ribose (400 MHz), CDCl₃.

FIG. 6 illustrates the chemical assignment of the ¹H NMR spectrumcompound 6 as shown in FIG. 6.

FIG. 7 illustrates a prior art biosynthetic incorporation of KDO intolipopolysaccharides and a prior art enzymatic synthetic route employingKDO-8 phosphate synthase and KDO-8-P phosphatase as catalysts andarabinose-5-P and PEP as substrates.

FIGS. 8A-I illustrate a synthetic scheme employing an aldolasecondensation reaction and an excess of pyruvate for producing KDO from avariety of starting sugars.

FIG. 8A illustrates a synthetic scheme employing KDO aldolase andD-arabinose.

FIG. 8B illustrates a synthetic scheme employing KDO aldolase andD-ribose.

FIG. 8C illustrates a synthetic scheme employing KDO aldolase and2-deoxy-D-ribose.

FIG. 8D illustrates a synthetic scheme employing KDO aldolase andD-erythrose.

FIG. 8E illustrates a synthetic scheme employing KDO aldolase andD-glyceraldehyde.

FIG. 8F illustrates a synthetic scheme employing KDO aldolase andD-threose.

FIG. 8G illustrates a synthetic scheme employing KDO aldolase andL-glyceraldehyde.

FIG. 8H illustrates a synthetic scheme employing KDO aldolase andL-mannose.

FIG. 8I illustrates a synthetic scheme employing sialic acid aldolaseand D-mannose.

FIGS. 9A and B illustrate a synthetic route employing a decarboxylationof KDO aldolase condensation products. Additionally, FIG. 9A illustratesthe stabilization of a planar conformer of the radical intermediate,stabilized both by the electron-donating and withdrawing effects,thereby allowing the maximum interaction between the one-electron porbital and the lone pair electrons on the adjacent ring oxygen.

DETAILED DESCRIPTION

A New Source of KDO Aldolase

Aureobacterium barkerei strain KDO-37-2 (ATCC 49977) was isolated fromgarden soil using KDO as a major carbon source. The microorganism(strain KDO-37-2) grows well on LB medium. It is aerobic, gram-positive,not motile and with colonies 1-3 millimeters in diameter on LB agarplates. The colony morphology is circular, low convex, entire edge andproduces yellow pigment. Optimum growth temperature is about 30° C.Major fatty acids are anteiso-C_(15:0) and anteiso-C_(17:0). The strainwas identified as Aureobacterium barkerei according to Bergey's manual.A deposit of Aureobacterium barkerei strain KDO-37-2 was made Jul. 30,1992 with the American Type Culture Collection (ATCC) 12301 ParklawnDrive, Rockville, Md., USA 20852 and was given Accession Number ATCC49977.

The deposit with accession Number ATCC 49977 was made in compliance withthe Budapest Treaty requirements that the duration of the depositsshould be for 30 years from the date of deposit at the depository or forthe enforceable life of a U.S. patent that matures from thisapplication, whichever is longer. The cell line will be replenishedshould it become non-viable at the depository.

A preferred medium for KDO aldolase production is defined as follows:NH₄Cl (5 grams), K₂SO₄ (1 gram), MgSO₄.7H₂O (200 milligrams), CaCl₂ (20milligrams), FeSO₄.7H₂O (1 milligram), yeast extract (1 gram),Na₂HPO₄.7H₂O and KH₂PO₄ (3 grams) in distilled water (1 liter) at pH7.2. A seed culture may be made by admixing in a 100 milliliterErlenmeyer flask 50 milliliters of the above medium together with 25microliters of a 40% glucose solution and 100 milligrams (0.2%) of KDO.The seed culture is then inoculated with a loopful of Aureobacteriumbarkerei strain KDO-37-2 (ATCC 49977). The flask is then shaken at 250r.p.m. on a gyrorotory shaker at 30° C. for 16 hours. The seed culturethus obtained may then be poured into the 1950 milliliters of the samemedium containing LDO as a major carbon source. The culture wasincubated for 24 hours at 30° C. with shaking. The cells may beharvested as a source of KDO aldolase enzyme.

For routine culture preservation, the culture can grow on LB medium andcan be incubated overnight at 30° C. This strain of Aureobacteriumbarkerei is shown to be a source of KDO aldolase (EC 4.1.2.23) having abroad substrate specificity with respect to the reverse aldolcondensation reaction.

A New Source of KDO Aldolase

KDO aldolase (EC 4.1.2.23) was first reported by Ghalambor and Heath in1966 as the enzyme responsible for the KDO degradation (FIG. 7). Aftertheir preliminary investigation on the substrate specificity as well asthe μmol scale synthesis of KDO, no synthetic application of this enzymehas been reported, while the related enzyme N-acetylneuraminic acid(sialic acid) aldolase has been extensively studied.

It is disclosed herein that the Gram-positive bacterium Aureobacteriumbarkerei strain KDO-37-2 can be induced to contain high levels of KDOaldolase. The aldolase activity from this source was assayed accordingto Aminoff's method (Biochem. J. 1961, 81, 384). Two liters of culturecontained 10.2 U based on the degradation of KDO. This KDO activity is 4times and 8 times higher than the corresponding KDO activity fromEscherichia coli K-12 and Aerobacter cloacae, respectively, as reportedby Gharambor (supra).

Partially purified KDO aldolase simply obtained by ammonium sulfateprecipitation (8.0 U/mL; 0.19 U/mg for degradation of KDO) was used insubstrate-specificity studies reported herein. The KDO aldolase employedfor the kinetic analysis reported herein, was further purified via DEAEsepharose and phenyl sepharose column chromatography to a specificactivity of 5.7 U/mg. The K_(m) for D-arabinose and V_(max) are 1.2 Mand 0.73 U/mg, respectively. The unusually high concentration of K_(m)in the condensation compared with that in the course of degradation(6×10⁻³ M for KDO) indicates that the enzyme may accept the open form ofaldoses as acceptors in the aldol condensation. The enzymatic reactionfavors the cleavage of KDO, with the equilibrium constantK_(eq)=[pyruvate][arabinose]/KDO=9×10⁻²M.

Substrate Specificity

This enzyme exhibits a wide substrate specificity. Several 3-6 carbonsugars were accepted as substrates for the condensation. From theresults shown in Table I and FIG. 1, the structural requirements of thesugar for this enzyme are as follows. At C-2 position, although thealdolase prefers an S configuration, the difference is not significant[examples: between L- and D-glyceraldehyde; D-threose and D-erythrose;D-arabinose, D-ribose and 2-deoxy-D-ribose]. It is noteworthy that thisenzyme also accepts D-ribose as a good substrate (rel. V=72%), whilethat from E. coli or Aerobacter cloacae poorly accepts this substrate(rel. V<5%), according to Gharambor (supra). At C-3 position, thisenzyme prefers an R-configuration [examples: comparison betweenD-arabinose and L-arabinose, D-lyxose and D-xylose]. Hexoses aregenerally not as good substrates as tetroses and pentoses, even in thecase of D-altrose (rel. V=25%) and L-fucose (rate not detectable), bothbeing homoanalogs of the natural substrate D-arabinose. The reason thatL-mannose is a better substrate than D-mannose is because the former hasthe favorable 2R,3R configurations and the latter has the unfavorable2S,3S configurations. Finally, neither fluoropyruvate nor ketohexose wasaccepted by this enzyme.

The Aldol Condensation

The enzymatic synthesis of KDO on multi-mmol scales using 10 molarexcess of pyruvate worked well (e.g. 1 was obtained in 67% yield). Thesynthetic route is illustrated in FIG. 8A. The reagents employed in thissynthesis are as follows:

Step Reagent or Enzyme (a) KDO aldolase (b) Ac₂O/py, DMAP (c) CH₂N₂.

The yield of the enzymatic reaction is comparable to the highest oneobtained by the modified Cornforth synthesis (66%). The crystalline KDOammonium salt monohydrate was isolated in 37% yield: [α]²⁶D +40.3° (c2.06, H₂O) [lit. according to Unger: [α]²⁷D +42.3° (c, 1.7, H₂O),authentic sample from Sigma [α]²⁶D +40.2° (c 2.06, H₂O)]. (Unger: Adv.Carbohydr. Chem. Biochem. 1981, 38, 323.) The ¹H NMR spectrum in D₂O isidentical with that of an authentic sample, although it is complicatedby the fact that KDO exists as an anomeric mixture of pyranose andfuranose forms, and readily cyclizes to the corresponding lactone inaqueous solution. The crystalline ammonium salt was further converted topentaacetate methyl ester derivative 2, whose ¹H NMR spectrum was ingood accordance with that reported previously and clearly shows the⁵C₂-pyranose conformation (Table II).

Several substrates with good or fair relative rate are shown to beemployable in the aldol condensation. The reactions with D-ribose and2-deoxy-D-ribose are illustrated in FIGS. 8B and 8C respectively. Thesereactions took place smoothly to give 3 (57% after derivation to 4) and5 (47% as 6), respectively. ¹H NMR spectra of 3, 4, 5, and 6 clearlyshow a ⁵C₂ pyranose form in both products (Table III). The ¹H spectrumof 6 is shown in FIG. 2. It is noteworthy that in these cases, eventhough the relative rates are lower (72% for D-ribose and 71% for2-deoxy-D-ribose) than that of D-arabinose, TLC analysis of the reactionproducts showed no starting material left, whereas a substantial amountof starting material always remains in the reaction with D-arabinose. Itis suggested that formation of the pyranose form of 3 and 5, where allsubstituents are located in the stable orientation, further shifted theequilibrium toward condensation.

The products 7 (3-deoxy-D-arabino-2-heptulosonic acid, DAH, 39% as 8)and 9 (11% as 10) were also obtained from D-erythrose andD-glyceraldehyde, as illustrated in FIGS. 8D and 8E respectively. Theseyields indicate that this aldolase-catalyzed condensation is also usefulfor the synthesis of lower homologs of KDO. The phosphate of 7 (DAHP)plays an important role in the shikimate synthesis pathway in plants andmicroorganisms. The selected chemical shifts and coupling constants forthe ¹H NMR spectra of products 3-10 are summarized in Table III.

FIG. 8F illustrates the aldolase catalyzed condensation reaction can beemployed to produce product 11 from D-threose. Product 11 has a ¹H NMRspectrum similar to that of KDO. The reaction with L-glyceraldehyde,illustrated in FIG. 8G afforded 13 (2-keto-3-deoxy-L-gluconic acid,KDG), an enantiomer of D-KDG, whose phosphate (KDGP) is an intermediatein the Entner-Doudoroff pathway. (Entner, N.; Doudoroff, M. J. Biol.Chem. 1952, 196, 853.) The ¹H NMR spectrum of 13 was very complicated(see experimental). To clarify the stereochemistry, preparation ofderivatives was attempted; however, the products were still difficult toidentify. The only isolable component from 11 was a bicyclic lactone.The structure was determined as 12 (FIG. 8F) by comparing its ¹H NMRspectrum with that of the higher homolog 12′, which had been obtainedfrom KDO and unambiguously characterized previously. (Charon, D.;Auzanneau, F.-I.; Mérienne, C.; Szabó, L. Tetrahedron Lett. 1987, 23,1393.)

In its ¹H NMR spectrum (Table II), a long range coupling between H-3 andH-5 (0.6 Hz) indicates that the pyranose form of the product exists as atwisted boat conformation, and all of the coupling constants areconsistent with those observed in the case of 12′. It is interestingthat in the spectra of 11, 13 and KDO, a substantial proportion of thesimilar signals were observed, where one of the H-3 signal appears atvery low field (Table II). From these results, it is assumed that thebicyclic 1<<5 lactones 1′, 11′ and 13′ form at nearly neutral pH. Theformation of 1<<7 lactone is excluded, since those signals were observedin the case of a hexulosonate 13′ without any C-7 hydroxy group. Thehomologs prepared here also proceed through a spontaneous 1<<5 lactoneformation, as already proposed previously for KDO. (Menton, L. D., etal. Carbohydr. Res. 1980, 80, 295.) Compound 13 mainly exists as ⁵C₂pyranose form as indicated in 14.

The reaction with L-mannose illustrated in FIG. 8H gave 15(3-deoxy-L-glycero-L-galacto-2-nonulosonic acid, L-KDN, 61% as 16),which is an enantiomer of D-KDN, a component in polysialoglycoproteinand ganglioside of rainbow trout eggs. (Lin, C.-H., et al., J. Am. Chem.Soc., in press; Nadano, D. et al. J. Biol. Chem. 1986, 261, 11550; andSong, Y., et al. J. Biol. Chem. 1991, 266, 21929.) The optical rotation[[α]²⁵D+26.3° (CHCl₃) ] and 1H NMR spectrum of 16 were in goodaccordance with those of 16′ [[α]²⁵D −26.0° (CHCl₃)], which was obtainedvia reaction with D-mannose catalyzed by sialic acid aldolase, asillustrated in FIG. 8I, except for the sign of rotation (Tetrahedron1990, 46, 201). The availability of both enantiomers of KDN may developnew analogs of sialyl oligosaccharides. (Ichikawa, Y., et al. Anal.Biochem. 1992, 202, 215.)

Finally, the aldol reaction with an unnatural sugar containing afluorine atom was conducted to give 18 (19% of 19). By comparing the ¹HNMR spectra, the proportion of the β-isomer (10.71) of 18 was ca. 1.5times higher than that of KDO (6.9%), probably due to the absence offuranose and 1<<5 lactone forms. This result suggests that 18 might be agood substrate for CMP-KDO synthetase, since the enzyme accepts theunstable β-form of KDO as a substrate. (Kohlbrenner, W. E. and Fesik, S.W., J. Biol. Chem. 1985, 260, 14695.) We therefore synthesized 18 in alarger scale by combining the use of KDO aldolase and pyruvatedecarboxylase, which made the workup procedure much easier. Preliminarystudy using 18 toward CMP-KDO synthetase which had recently been clonedand over-expressed in this group showed that 18 was accepted to theenzyme.

Based on these results, the stereochemical course of the aldolcondensation catalyzed by this KDO aldolase is probably as follows: Theattack of pyruvate always takes place on the re face of the carbonylgroup of the substrates, a facial selection complementary to sialic acidaldolase reactions (si face attack). The stereochemical requirements ofsubstrates and the stereochemical course of the aldol condensation areindicated in FIG. 3. It is concluded that in general the enzyme acceptssubstrates with an R-configuration at C-3. The substrates with an Sconfiguration at C-2 is kinetically favored, while those with Rconfiguration at C-2 are thermodynamically favored to give a betteryield.

Synthesis of Decarboxylated Analogs

Decarboxylation of KDO and its analogs will yield the correspondingaldose derivatives. A synthetic route employing decarboxylation of KDOaldolase condensation products is illustrated in FIGS. 9A and 9B. Thereagents employed in these synthetic routes is as follows:

Step Reagent (a) Ac₂, DMAP/pyridine (b) CsCO₃, BnBr/DMF (c) H₂,Pd-C/EtOH (d) (COCl)₂/toluene (e) 22, DMAP/pyridine-toluene (f) t-BuSH,hν (g) Et₂NCH₂CH₂CH₂N═C═NEt.HCl (WSCl)-Cl, 22 (5 eq.), t-BuSH, DMAP,Et₃N, MS 4Å/CH₂Cl₂, hν

The aldodeoxyheptose structure is particularly interesting since anumber of heptoses are widely distributed in nature, some of which playimportant roles in metabolic pathways. Barton's radical-mediateddecarboxylation of the penta-O-acetyl derivative 20a obtained from thecorresponding benzyl ester 20b seems to be the most straightforwardroute to the desired heptose derivative 21. (e.g., Crich, D. and Lim, L.B. L. J. Chem. Soc. Perkin I 1991, 2209 and Auzanneau, F.-I. et al.Carbohydr. Res. 1990, 201, 337.)

There have recently been growing interests in the synthesis ofphysiologically active carbohydrate- and nucleic acid-related compoundsvia anomeric radical intermediates. It appears to us thatradical-mediated reaction stabilized both electron withdrawing anddonating group (capto-dative effect), e.g. Viehe, H. G. et al. Acc.Chem. Res. 1985, 18, 148.) at anomeric position [—C()(OAc)O-type] israre (only a few related examples [eg. —C()(CO₂Me)O-type,—C()(CHF₂)O-type] are known), while examples in the case of simpleanomeric radical [—C()(H or R)O-type] and the one bearing two electrondonating oxygen atom [—C()(OR))-type] have been extensively studied.(e.g. Crich, D. and Lim, L. B. L. J. Chem. Soc. Perkin I 1991, 2205 andJ. Chem. Soc. Perkin I 1991, 2209; Schmidt, R. et al. Tetrahedron Lett.1988, 29, 3643; Myrvold, S. et al. J. Am. Chem. Soc. 1989, 111, 1861;Motherwell, W. B. et al. Synlett. 1989, 68; and Samadi, M. J. Med. Chem.1992, 35, 63.) The radical intermediate was formed by the thermaldecomposition of the thiohydroxamate 20c generated in situ from thecorresponding acid chloride and 22 in the presence ofazobisisobutyronitrile (AIBN). The subsequent trapping with tributyltinhydride resulted in only a disappointing (less than 2%) yield of 21. Theyield was, however, dramatically improved to 68% by irradiation withwhite light in the presence of t-butylmercaptane.

The ¹H NMR spectrum of 21 clearly shows the exclusive β-anomer (δ5.75,dd, J_(1.2eq)=3.0, J_(1.2ax)=10.0 Hz, H-1), indicating that theabstraction of hydrogen atom from t-butylmercaptane took place at thebottom side of the six-membered ring. The proposed mechanism for theexclusive formation of β-isomer is as follows. The stable conformer ofthe radical intermediate which is stabilized both by theelectron-donating and withdrawing effects is supposed to be in a planeform as depicted in FIGS. 9A and 9B, which allows the maximuminteraction between the one-electron p orbital and the lone pairelectrons on the adjacent ring oxygen. t-Butylmercaptane is easilyaccessible from the bottom side, while the approach from the top side issterically hindered by the hydrogen and acetoxy groups. This explanationin terms of kinetic control is well matched with the thermodynamicstability of the β-product.

The radical process was also applied to the synthesis of thedecarboxylated analog of N-acetylneuraminic acid. It turned out,however, that all attempts for the synthesis of the acyl chlorideresulted in a complex mixture, even from fully protected peracetate form23a of sialic acid, because NHAc proton still has a substantialreactivity to chlorinating reagents. The direct formation ofthiohydroxamate 23b was also found to be difficult because of theinherent steric hindrance around carbonyl group in the startingmaterial. Through an extensive examination of the reaction conditions,it was found that the combination of ethyl(diethylamino)pronylcarbodiimide hydrochloride (WSCI-Cl, 1.5 eq) andexcess of 22 (5.0 eq) worked well for the in situ formation anddegradation of thiohydroxamate, to give 24 (27% yield from 23a). Thiscondition has the advantage that the reaction can be carried out in onestep. The newly formed product was exclusively an α-anomer where the OAcgroup is located in the equatorial orientation, consistent with theresult obtained in the decarboxylation of KDO derivative. (Haverkamp, J.et al. Eur. J. Biochem. 1982, 122, 305.)

PREPARATION OF EXAMPLES

General

Optical rotations were measured on Perkin-Elmer 241 spectrophotometer UVand visible spectra were recorded on a Beckmann DU-70 spectrometer. ¹Hand ¹³C NMR spectra were recorded at 400 and 500 MHz on Bruker AMX-400and AMX-500 spectrometer. High-resolution mass spectra (HRMS) wererecorded on a VG ZAB-ZSE mass spectrometer under fast atom bombardment(FAB) conditions. Column chromatography was carried out with silica gelof 70-230 mesh. Preparative TLC was carried out on Merck Art. 5744 (0.5mm).

Isolation of the Microorganism

Aureobacteriur barkerei containing high levels of KDO aldolases wasselected with the S medium containing 0.25% of synthetic KDO mixture ascarbon source (20 mL) in serum bottles (158 mL) and incubated at 37° C.for 2 days with shaking (250 r.p.m.). (McNicholas, P. A. et al.Carbohydr. Res. 1986, 146, 219 and Shirai, R.; and Ogura, H. TetrahedronLett. 1989, 30, 2263.) The bottles which showed turbidity weretransferred to the same fresh medium. After several transfers, thecultures were plated on the S medium agar plates (1.5% agar) containing0.25% of synthetic KDO mixture. The isolated colonies were transferredto the liquid medium as described above. To confirm the utilization ofKDO, the disappearance in the medium was monitored by TLC as describedin the synthesis of KDO. The cultures which showed the utilization ofKDO were harvested by centrifugation and resuspended in 50 mM phosphatebuffer (pH 7.0). The cell suspension was incubated with 1% (w/v) ofauthentic KDO (from Sigma) at 37° C. overnight to confirm thedegradation of KDO by TLC. The cultures were then replated on LB agarplates to ensure the purity of the culture.

Preparation of the Enzyme

With one slight modification, the incubation was carried out accordingto the procedure reported by Gharambor (supra). The ingredients of themedium were as follows: NH₄Cl (5 g), K₂SO₄ (1 g), MgSO₄.7H₂O (200 mg),CaCl₂ (20 mg), FeSO₄.7H₂O (1 mg), yeast extract (1 g), Na₂HPO₄.7H₂O (10g), and KH₂PO₄ (3 g) in distilled water (1 L), at pH 7.2. To a 50 mL ofthis medium in a 100 mL Erlenmeyer flask, were added D-glucose (40%solution in water, 25 μL) and KDO (100 mg, 0.2%), and a loopful ofAureobacterium barkerei KDO-37-2 was incolutated. The flask was shakenat 250 r.p.m. on a gyrorotary shaker at 30° C. for 16 h. The seedculture thus obtained was poured into the 1950 mL of the same incubationmedium containing KDO (3.9 g). The mixture was divided and poured intotwo of 2.8 L Erlenmeyer flasks. The flasks were shaken at 250 r.p.m. at30° C. for 24 h. The growth of microorganism was estimated by OD at 600nm to be 1.90. The cells were harvested at 10,000×g for 30 min at 4° C.and washed with 50 mM potassium-sodium phosphate buffer (pH 7.5). Thecollected cells were then resuspended in the same buffer solution (20mL) and disrupted by French-pressure apparatus (at 16,000 lb/in). Thecell debris were removed by centrifuge at 23,000×g for 1 h at 4° C. togive the supernatant (ca. 20 mL) as the crude enzyme preparation. Theenzyme activity was determined to be 1.45 U/mL for the degradation ofKDO according to the method of Aminoff (Biochem. J. 1961, 81, 384).Ammonium sulfate precipitation between 45-75% saturation was collectedand dialyzed in phosphate buffer (2 L; 100 mM, 1 mM of dithiothreitol, 2L) to give partially purified enzyme (13.5 mL, 1.73 U/mL for KDOdegradation), according to the method of Kim (J. Am. Chem. Soc. 1988,110, 6481)

Kinetic Measurements

The rates for aldolase-catalyzed reactions were obtained by measuringthe amount of remaining pyruvate, according the method of Kim (supra).The reactions were carried out in 0.1 M phosphate buffer (pH 7.5)containing: varied concentrations of pyruvate, 2.0, 3.33, 5, and 10 mM;varied concentrations of D-arabinose, 0.2, 0.25, 0.33, and 0.50 M in 0.5mL of solution. Each solution was incubated at 37° C. Periodically, asmall aliquot (25-100 μL) was withdrawn and mixed with an assay solution(1.4 mL) containing 0.1 M phosphate (pH 7.5) buffer, 0.3 mM NADH, and20-30 U of L-lactate dehydrogenase. The decrease in absorbance at 340 nmwas measured and converted into the amount of the unreacted pyruvateusing 6220 M⁻¹cm⁻¹ for the molecular absorbance of NADH. The kineticparameters were obtained from the Lineweaver-Burk plots.

For the relative rate measurements, the concentration of pyruvate(fluoropyruvate) and sugar were fixed at 10 mM and 0.5 M, respectively.Other conditions were the same as above.

Example 1 Ammonium 3-deoxy-α-D-manno-2-octulosonate monohydrate (KDOazmonium salt monohydrate, 1)

D-Arabinose (250 mg, 1.67 mmol), sodium pyruvate (1.83 g, 16.7 mmol),dithiothreitol (1.5 mg), NaN₃ (2% solution in water, 100 μL),NaHPO₄.7H₂O (53 mg), and KH₂PO₄ (13 mg) were added to the KDO aldolase(5.1 U, 10 mL). The pH was adjusted to 7.5 and the mixture was stirredunder N₂ at 30° C. for 3 days. The product was purified by treatmentwith a Dowex-1 resin column (bicarbonate form) eluted with a lineargradient from 0 to 0.25 M of ammonium bicarbonate. KDO ammonium salt wasfurther purified by Biogel P-2 column. The fraction eluted with H₂Ocontaining KDO was collected and its total amount was estimated to be1.11 mmol (67%) by Aminoff's assay (supra). The residue afterlyophilization was recrystallized from aqueous ethanol to give colorlessplates (168 mg, 37% from D-arabinose): mp 123-125° C. (decomposition)[lit. according to Hershberger: mp 121-123° C., authentic sample fromSigma mp 123-125° C. (decomposition)]; [α]²⁶D +40.3° (c 2.06, water)[lit. according to Hershberger: [α]²⁷D +42.3° (c 1.7, water), authenticsample from Sigma [α]²⁶D +40.2° (c 2.03, water)]. Its ¹H NMR spectrum inD₂O was identical with that of an authentic sample. (Hershberger: J.Biol. Chem. 1968, 243, 1585.) A small portion was converted topentaacetate methyl ester derivative 2: ¹H NMR (CDCl₃) δ1.994 (3H, s,acetyl), 1.998 (3H, s, acetyl), 2.045 (3H, s acetyl), 2.108 (3H, s,acetyl), 2.139 (3H, s, acetyl), 2.201 (1H, dd, J_(3ax,4)=12.0,J_(3ax,3eq)=13.0 Hz, H-3ax), 2.245 (1H, dd, J_(3eq,4)=6.0,J_(3eq,3ax)=13.0 Hz, H-3eq), 3.810 (3H, s, COOCH₃), 4.113 (1H, dd,J_(8′,) 7 =12.5, J_(8′,) 8 =12.5 Hz, H-8′), 4.173 (1H, dd, J_(6.5)=1.3,J_(6,7)=9.5 Hz, H-6), 4.475 (1H, dd, J_(8,7)=4.0, J_(8,8′)=12.5 Hz,H-8), 5.220 (1H, ddd, J_(7,8)=4.0, J_(7,6)=9.5, J_(7,8′)=12.5 Hz, H-7),5.322 (1H, ddd, J_(4,5)=3.0, J_(4,3eq)=6.0, J_(4,3eq)=6.0,J_(4,3ax)=12.0 Hz, H-4), 5.385 (1H, dd, J_(5,6)=1.3, J_(5,4)=3.0 Hz,H-5). The ¹H NMR spectrum was in good accordance with that reportedpreviously by Unger (Adv. Carbohydr. Chem. Biochem. 1981, 38, 323).

Example 2 Methyl2,4,5,7,8-penta-O-acetyl-3-deoxy-α-D-altro-2-octulosonate (4)

In the same manner as described for the preparation of 1, the product 3(as ammonium salt) was prepared from D-ribose (0.33 mmol): ¹H NMR (D₂O)δ1.773 (1H, dd, J_(3ax,4)=11.9, J_(3ax,3eq)=13.0 Hz, H-3ax), 2.148 (1H,dd, J_(3eq,4)=5.1, J_(3eq,3ax)=13.0 Hz, H-3eq), 3.500 (1H, dd,J_(5,4)=9.1, J_(5,6)=10.0 Hz, H-5), 3.745 (1H, dd, J_(8,7)=7.3,J_(8,8′)=12.1 Hz, H-8), 3.789 (1H, dd, J_(8′,7)=3.7, J_(8′,8)=12.1 Hz,H-8′), 3.809 (1H, dd, J_(6,7)=2.8, J_(6,5)=10.0 Hz, H-6), 3.901 (1H,ddd, J_(4,3eq)=5.1, J_(4,5)=9.1, J_(4,3ax)=11.9 Ha, H-4), 4.004 (1H, dd,J_(7,6)=2.8, J_(7,8′)=3.7, J_(7,8)=7.3 Hz, H-7). This was converted to 4by the successive treatment with acetic anhydride-pyridine-DMAP (seealso the preparation of 20b) and etherial diazomethane solution. Theproduct was purified with silica gel preparative TLC to afford 4 (87.7mg, 57% from D-ribose) as an oil, [α]²⁵D +70.9° (c 0.81, CHCl₃); ¹H NMR(CDCl₃) δ2.010 (1H, dd, J_(3ax,4)=11.6, J_(3ax,3eq)=13.5 Hz, H-3ax),2.030 (3H, s, acetyl), 2.050 (3H, s, acetyl), 2.064 (3H, s, acetyl),2.105 (3H, s, acetyl), 2.154 (3H, s, acetyl), 2.559 (1H, dd,J_(3eq,4)=5.2, J_(3eq,3ax)=13.5 Hz, H-3eq), 3.793 (3H, s, COOCH₃), 4.084(1H, dd, J_(6,7)=3.2, J_(6,5)=10.3 Hz, H-6), 4.241 (1H, dd, J_(8,7)=7.0,J_(8,8′)=12.0 Hz, H-8), 4.415 (1H, dd, J_(8′,7)=4.0 J_(8′,8)=12.0 Hz,H-8′), 5.110 (1H, dd, J_(5,4)=9.3, J_(5,6)=10.3 Hz, H-5), 5.169 (1H,ddd, J_(7,6)=3.2, J_(7,8′)=4.0, J_(7,8)=7.0 Hz, H-7), 5.271 (1H, ddd,J_(4,3eq)=5.2, J_(4,5)=9.3, J_(4,3ax)=11.6 Hz, H-4); ¹³C NMR (CDCl₃)δ20.52, 20.56, 20.56, 20.67, 20.67, 35.47, 53.12, 61.23, 68.33, 68.96,69.85, 71.98, 96.66, 166.21, 167.94, 169.52, 169.85, 169.89, 170.38.HRMS (M+Cs⁺) calcd C₁₉H₂₆O₁₃Cs 595.0428, found 595.0428.

Example 3 Methyl2,4,7,8-tetra-O-acetyl-3,5-dideoxy-α-D-manno-2-octulosonate (6)

In the same manner as 3, the product 5 (as ammonium salt) was preparedfrom 2-deoxy-D-ribose (0.33 mmol): ¹H NMR (D₂O) δ1.400 (1H, ddd,J_(5ax,4)=11.9, J_(5ax,6)=11.9, J_(5ax,5eq)=12.3 Hz, H-5ax), 1.591 (1H,dd, J_(3ax,4)=12.1, J_(3ax,3eq)=12.7 Hz, H-3ax), 2.009 (1H, dddd,J_(5eq,3eq)=1.8, J_(5eq,6)=2.2, J_(5eq,4)=4.6, J_(5eq,5ax)=12.3 Hz,H-5eq) 2.094 (1H, ddd, J_(3eq,5eq)=1.8, J_(3eq,4)=4.6, J_(3eq,3ax)=12.7Hz, H-3eq), 3.398 (1H, dd, J_(8,7)=7.1, J_(8,8′)=11.8 Hz, H-8), 3.588(1H, dd, J_(8′,7)=4.1, J_(8′,8)=11.8 Hz, H-8′), 3.786 (1H, ddd,J_(7,8′)=4.1, J_(7,6)=4.6, J_(7,8)=7.1 H8, H-7), 3.945 (1H, ddd,J_(6,5eq)=2.2, J_(6,7)=4.6, J_(6,5ax)=11.9 Hz, H-6), 4.112 (1H, dddd,J_(4,3eq)=4.6, J_(4,5eq)=4.6, J_(4,5ax)=11.9, J_(4,3ax)=12.1 Hz, H-4).This was converted to 6 (62.2 mg, 47% from 2-deoxy-D-ribose): [α]²⁵D+86.0° (c 0.56, CHCl₃); ¹H NMR (CDCl₃) δ1.488 (1H, ddd, J_(5ax,4)=12.0,J_(5ax,6)=12.0, J_(5ax,5eq)=12.7 Hz, H-5ax), 1.783 (1H, dd,J_(3ax,4)=11.6, J_(3ax,3eq)=13.1 Hz, H-3ax), 2.045 (3H, s, acetyl),2.054 (3H, s, acetyl), 2.070 (3H, s, acetyl), 2.123 (3H, s, acetyl),2.177 (1H, dddd, J_(5eq,3eq)=1.8, J_(5eq,6)=2.2, J_(5eq,4)=4.7,J_(5eq,5ax)=12.7 Hz, H-5eq), 2.454 (1H, ddd, J_(3eq,5eq)=1.8,J_(3eq,4)=4.8, J_(3eq,3ax)=13.1 Hz, H-3eq), 3.782 (3H, s, COOCH₃), 4.034(1H, ddd, J_(6,5eq)=2.2, J_(6,7)=7.6, J_(6,5ax)=12.0 Hz, H-6), 4.169(1H, dd J_(8,7)=5.1, J_(8,8′)=12.2 Hz, H-8), 4.457 (1H, dd,J_(8′,7)=2.8, J_(8′,8)=12.2 Hz, H-8′), 5.093 (1H, ddd, J_(7,8′)=2.8,J_(7,8)=5.1, J_(7,6)=7.6 Hz, H-7), 5.186 (1H, dddd, J_(4,5eq)=4.7,J_(4,3eq)=4.8, J_(4,3ax)=11.6, J_(4,5ax)=12.0 Hz, H-4); ¹³C NMR (CDCl₃)δ20.56, 20.56, 20.73, 20.96, 32.21, 36.03, 52.96, 61.82, 65.72, 69.00,71.96, 97.61, 167.02, 167.96, 169.81, 170.06, 170.32. HRMS (M+Cs⁺) calcdC₁₇H₂₄O₁₁Cs 537.0373, found 537.0373.

Example 4 Methyl2,4,5,7-tetra-O-acetyl-3-deoxy-α-D-arabino-2-heptulosonate (8)

7: ¹H NMR (D₂O) δ1.773 (1H, dd, J_(3ax,4)=11.8, J_(3ax,3eq)=13.0 Hz,H-3ax), 2.180 (1H, dd, J_(3eq,4)=5.1, J_(3eq,3ax)=13.0 Hz, H-3eq), 3.433(1H, dd, J_(5,4)=9.2, J_(5,6)=9.5 Hz, H-5), 3.744 (1H, ddd, J_(6,7)=3.5,J_(6,7′)=3.5, J_(6,5)=9.5 Hz, H-6) 3.807 (1H, m, H-7), 3.812 (1H, m,H-7′), 3.930 (1H, ddd, J_(4,3eq)=5.1, J_(4,5)=9.2, J_(4,3a)=11.8 Hz,H-4).

8: (50.0 mg, 39% from 0.33 mmol of D-erythrose): [α]²⁵D +54.0° (c 0.50,CHCl₃); ¹H NMR (CDCl₃) δ2.034 (3H, s, acetyl), 2.053 (3H, s, acetyl),2.087 (3H, s, acetyl), 2.087 (1H, dd, J_(3ax,4)=11.4, J_(3ax,3eq)=13.6Hz, H-3ax), 2.173 (3H, s, acetyl), 2.658 (1H, dd, J_(3eq,4)=5.2,J_(3eq,3ax)=13.6 Hz, H-3eq) 3.808 (3H, s, COOCH₃) 4.058 (1H, dd,J_(6,7)=2.3, J_(6,7′)=4.3, J_(6,5)=10.2 Hz, H-6), 4.100 (1H,J_(7,6)=2.3, J_(7,7′)=12.4 Hz, H-7), 4.355 (1H, J_(7′,6)=4.3,J_(7′,7)=12.4 Hz; H-7′); ¹³C NMR (CDCl₃) δ20.65, 20.76, 20.76, 20.84,35.58, 53.31, 61.69, 68.16, 68.37, 71.51, 97.29, 166.41, 168.43, 169.61,170.13, 170.77. HRMS (M+Cs⁺) calcd C₁₆H₂₂O₁₁Cs 523.0216, found 523.0216.

Example 5 Methyl 2,4,5-tri-O-acetyl-2-keto-3-deoxy-α-D-galactonate (10)

9: ¹H NMR (D₂O) δ1.795 (1H, dd, J_(3ax,4)=11.6, J_(3ax,3eq)=13.1 Hz,H-3ax), 2.176 (1H, dd, J_(3eq,4)=5.1, J_(3eq,3ax)=13.1 Hz, H-3eq),3.60-3.65 (2H, m), 3.77-3.91 (2H, m).

10: (11.0 mg, 11% from 0.33 mmol of D-glyceraldehyde): [α]²⁵D +31.8° (c1.10, CHCl₃); ¹H NMR (CDCl₃) δ1.948 (1H, dd, J_(3ax,4)=11.2,J_(3ax,3eq)=13.5 Hz, H-3ax), 2.055 (3H, s, acetyl), 2.059 (3H, s,acetyl), 2.170 (3H, s, acetyl), 2.618 (1H, dd, J_(3eq,4)=5.2,J_(3eq,3ax)=13.5 Hz, H-3eq), 3.629 (1H, dd, J_(6ax,5)=10.6,J_(6ax,6eq)=11.3 Hz, H-6ax), 3.809 (3H, s, COOCH₃), 4.149 (1H, dd,J_(6eq,5)=5.7, J_(6eq,6ax)=11.3 Hz, H-6eq), 5.049 (1H, ddd,J_(5,6eq)=5.7, J_(5,4)=9.5, J_(5,6ax)=10.6 Hz, H-5), 5.320 (1H, ddd,J_(4,3eq)=5.2, J_(4,5)=9.5, J_(4,3ax)=11.2 Hz, H-4); ¹³C NMR (CDCl₃)δ20.67, 20.72, 20.89, 35.81, 53.25, 62.17, 67.66, 68.49, 96.80, 166.96,168.50, 169.84, 170.05. HRMS (M+Cs⁺) calcd C₁₃H₁₈O₉Cs 451.0005, found451.0005.

Example 6 2,4,7-Tri-O-acetyl-3-deoxy-α-D-lyxo-2-heptulosonic acid 1<<5lactone (12)

11: ¹H NMR (D₂O) δ1.90-1.98 (m, H-3 of the major component); a minorpair of H-3 protons: 2.072 (dd, J_(3,4)=3.1, J_(3,3′)=14.2 Hz, H-3),2.576 (dd, J_(3′,4)=7.3, J_(3′,3)=14.2, Hz, H-3′); another minor pair ofH-3 protons: 2.301 (dd, J=7.0, 13.4 Hz), 2.384 (dd, J=7.0, 13.4 Hz);3.60-3.95 (m), 3.95-4.20 (m), 4.48-4.52 (m).

12: (1.9 mg): ¹H NMR (CDCl₃) δ2.096 (3H, s, acetyl), 2.127 (3H, s,acetyl), 2.180 (3H, s, acetyl), 2.339 (1H, ddd, J_(3,5)=0.6,J_(3,4)=2.4, J_(3,3′)=14.9 Hz, H-3), 2.972 (1H, dd, J_(3′,4)=9.4,J_(3′,3)=14.9 Hz, H-3′), 4.180 (1H, ABX type, J_(6,7)=5.6, J_(6,7′)=9.9Hz, H-6), 4.28-4.35 (2H, m, ABX type, H-7, H-7′), 4.904 (1H, d,J_(5,4)=2.0 Hz, H-5), 5.164 (1H, ddd, J_(4,5)=2.0, J_(4,3)=2.4,J_(4,3′)=9.4 Hz, H-4). HRMS (M+Cs⁺) calcd C₁₃H₁₆O₉Cs 448.9849, found448.9858.

Example 7 Methyl 2,4,5-tri-O-acetyl-2-keto-3-deoxy-α-L-gluconate (14)

13 (L-KDG): ¹H NMR (D₂O) A major pair of H-3 protons: δ1.873 (dd,J_(3eq,4)=5.2, J_(3eq,3ax)=13.0 Hz, H-3eq), 1.984 (dd, J_(3ax,4)=11.9,J_(3ax,3eq)=13.0 Hz, H-3ax); a minor pair of H-3 protons: 2.051 (dd,J_(3,4)=3.2, J_(3,3′)=14.1 Hz, H-3), 2.521 (dd, J_(3′,4)=7.5,J_(3′,3)=14.1 Hz, H-3′); a minor H-3 proton (²C₅ β-pyranose form issuggested): 2.167 (dd, J_(3,4)=4.0, J_(3,3′)=13.7 Hz), in this case theH-3′ proton could not be specified by overlapping of the signals;another minor pair of H-3 protons: 2.284 (dd, J=6.4, 13.1 Hz), 2.341(dd, J=6.4, 13.1 Hz); 3.60-4.10 (m), 4.15-4.20 (m), 4.30-4.40 (m).

14: (2.0 mg): ¹H NMR (CDCl₃) δ2.034 (3H, s, acetyl), 2.150 (3H, s,acetyl), 2.152 (3H, s, acetyl), 2.288 (1H, d, J_(3ax,4)=10.1 Hz, H-3ax),2.292 (1H, dd, J_(3eq,5)=0.4 Hz, J_(3eq,4)=7.0 Hz, H-3eq), 3.830 (3H, s,COOCH₃), 3.999 (1H, dd, J_(6eq,5)=1.5, J_(6eq,6ax)=13.2 Hz, H-6eq),4.092 (1H, dd, J_(6ax,5)=2.0, J_(6ax,6eq)=13.2 Hz, H-6ax), 5.251 (1H,dddd, J_(5,3eq)=0.4, J_(5,6eq)=1.5 J_(5,6ax)=2.0, J_(5,4)=2.7 Hz, H-5),5.313 (1H, ddd, J_(4,5)=2.7, J_(4,3eq)=7.0, J_(4,3ax)=10.1 Hz, H-4).HRMS (M+Na⁺) calcd C₁₃H₁₈O₉Na 341.0849, found 341.0849.

Example 8 Methyl2,4,5,7,8,9-hexa-O-acetyl-3-deoxy-β-L-glycero-L-galacto-nonulosonate(16)

15 (L-KDN): ¹H NMR (D₂O) δ1.773 (1H, dd, J_(3ax,4)=11.8,J_(3ax,3eq)=12.9 Hz, H-3ax), 2.168 (1H, dd, J_(3eq,4)=5.1,J_(3eq,3ax)=11.8 Hz, H-3eq), 3.579 (1H, dd, J_(5,4)=9.3, J_(5,6)=9.9 Hz,H-5), 3.654 (1H, dd, J_(9,8)=6.3, J_(9,9′)=11.8 Hz, H-9), 3.766 (1H,ddd, J_(8,9′)=2.6, J_(8,9)=6.3, J_(8,7)=9.0 Hz, H-8), 3.831 (1H, dd,J_(7,6)=1.1, J_(7,8)=9.0 Hz, H-7), 3.873 (1H, dd, J_(9′,8)=2.6,J_(9′,9)=11.8 Hz, H-9′), 3.925 (1H, dd, J_(6,7)=1.1, J_(6,5)=9.9 Hz,H-6), 3.971 (1H, ddd, J_(4,3eq)=5.1, J_(4,5)=9.3, J_(4,3ax)=11.8 Hz,H-4).

16 (108.3 mg, 61% from 0.33 mmol of L-mannose): [α]²⁵D +26.3° (c 1.14,CHCl₃); ¹H NMR (CDCl₃) δ2.084 (1H, dd, J_(3ax,4)=11.6, J_(3ax,3eq)=13.6Hz, H-3ax), 2.013 (3H, s, acetyl), 2.024 (3H, s, acetyl), 2.040 (3H, s,acetyl), 2.069 (3H, s, acetyl), 2.115 (3H, s, acetyl), 2.157 (3H, s,acetyl), 2.625 (1H, dd, J_(3eq,4)=5.3, J_(3eq,3ax)=13.6 Hz, H-3eq),3.790 (3H, s, COOCH₃), 4.141 (1H, dd, J_(9,8)=5.8, J_(9,9′)=12.6 Hz,H-9), 4.186 (1H, dd, J_(6,7)=2.3, J_(6,5)=10.3 Hz, H-6), 4.440 (1H, dd,J_(9′,8)=2.5, J_(9′,9)=12.6 Hz, H-9′), 4.975 (1H, dd, J_(5,4)=9.6,J_(5,6)=10.3 Hz, H-5), 5.150 (1H, ddd, J_(8,9′)=2.5, J_(8,9)=5.8,J_(8,7)=6.3 Hz, H-8), 5.264 (1H, ddd, J_(4,3eq)=5.3, J_(4,5)=9.6,J_(4,3ax)=11.6 Hz, H-4), 5.396 (1H, dd, J_(7,6)=2.3, J_(7,8)=6.3 Hz,H-7); ¹³C NMR (CDCl₃) δ20.46, 20.48, 20.58, 20.58, 20.67, 35.32, 53.06,61.67, 66.68, 67.21, 68.57, 70.00, 71.27, 97.14, 165.91, 168.03, 169.46,169.57, 169.80, 169.96, 170.41. HRMS (M+Cs⁺) calcd. C₂₂H₃₀O₁₅Cs667.0639, found 667.0639.

16′: [α]²⁵D −26.0° (c 1.00, CHCl₃). The ¹H NMR spectrum was identicalwith that of 16.

Example 9 2-Deoxy-2-fluoro-D-arabinose (17b)

To a solution of a tribenzoate 17a (available from Pfanstiehl Co., 500mg, 1.08 mmol) in ethanol (5 mL) was added 10 N NaOH aqueous solution(485 μL, 1.5 eq of each OBz group, total 4.5 eq) at room temperature.After 15 min, H₂O (10 mL) and ethanol (5 mL) were added and the mixturewas stirred and heated to 50° C. to dissolve the precipitated sodiumbenzoate. The mixture was further stirred for 1 h at room temperature.After ethanol was evaporated in vacuo, the residue was dissolved in H₂Oand Dowex 50W-X8 (H⁺ form) was added to acidify the mixture. Theprecipitated benzoic acid was filtered off, and the filtrate was treatedwith Dowex 1-X8 (HCO₃ ⁻ form) and filtered, then concentrated in vacuoto give 17b as colorless syrup (153 mg, 94%); ¹H NMR (D₂O) δ3.60-4.20(4H, m), 4.337 (ddd, J_(2,1)=7.7, J_(2,3)=9.3, J_(2,F)=51.8 Hz, H-2 ofβ-anomer), 4.666 (ddd, J_(2,1)=3.7 J_(2,3)=9.5, J_(2,F)=49.5 Hz, H-2 ofα-anomer), 4.763 (dd, J_(1,F)=3.3, J_(1,2)=7.7, H-1 of β-anomer), 5.434(dd, J_(1,F)=1.5, J_(1,2)=3.7 Hz, H-1 of α-anomer). This anomericmixture was used in the next step without further purification.

Example 10 Methyl2,4,7,8-tetra-O-acetyl-3,5-dideoxy-5-fluoro-α-D-manno-2-octulosonate(19)

18: ¹H NMR (D₂O) δ1.814 (dd, J_(3ax,3eq)=12.4, J_(3ax,4)=12.4 Hz, H-3axof β-anomer), 1.988 (1H, ddd, J_(3eq,5)=0.8, J_(3eq,4)=5.6,J_(3eq,3ax)=12.9 Hz, H-3eq of α-anomer), 2.058 (1H, dd, J_(3ax,4)=11.8,J_(3ax,3eq)=12.9 Hz, H-3ax of α-anomer), 2.461 (ddd, J_(3q,5)=0.8J_(3eq,4)=5.3, J_(3eq,3ax)=12.4 Hz, H-3eq of β-anomer), 3.663 (1H, dd,J_(8,7)=5.4, J_(8,8′)=12.1 Hz, H-8), 3.828 (1H, dd, J_(8′,7)=2.4,J_(8′,8)=12.1 Hz, H-8′), 3.80-3.95 (2H, m), 4.182 (1H, dddd,J_(4,5)=2.4, J_(4,3eq)=5.6, J_(4,3ax)=11.8, J_(4,F)=30.5 Hz, H-4), 4.957(1H, ddd, J_(5,3eq)=0.8 J_(5,4)=2.4, J_(5,F)=50.9 Hz, H-5).

19 (25.3 mg, 18% from 0.33 mmol of 17b): [α]²⁵D+96.4° (c 2.53, CHCl₃);¹H NMR (CDCl₃) δ2.043 (3H, s, acetyl), 2.067 (3H, s, acetyl), 2.131 (3H,s, acetyl), 2.137 (3H, s, acetyl), 2.271 (1H, dd, J_(3ax,4)=11.5,J_(3ax,3eq)=13.3 Hz, H-3ax), 2.319 (1H, dd, J_(3eq,4)=5.9,J_(3eq,3ax)=13.3 Hz, H-3eq), 3.805 (3H, s, COOCH₃), 4.073 (1H, dd,J_(6,7)=9.5, J_(6,F)=27.8 Hz, H-6), 4.154 (1H, dd, J_(8,7)=3.5,J_(8,8′)=12.5 Hz, H-8), 4.601 (1H, dd, J_(8′,7)=2.2, J_(8′,8)=12.5 Hz,H-8′), 4.827 (1H, dd, J_(5,4)=2.1, J_(5,F)=50.9 Hz, H-5), 5.240 (1H,dddd, J_(4,5)=2.1, J_(4,3eq)=5.9, J_(4,3ax)=11.5, J_(4,F)=21.3 Hz, H-4),5.288 (1H, ddd, J_(7,8′)=2.2, J_(7,8)=3.5, J_(7,6)=9.5 Hz, H-7); ¹³C NMR(CDCl₃) δ20.56, 20.56, 20.71, 20.83, 30.60, 53.18, 61.46, 66.45, (d,J_(C,F)=17.8 Hz), 67.89 (d, J_(C,F)=4.1 Hz), 69.60 (d, J_(C,F)=18.2 Hz,83.02 (d, J_(C,F)=186.2 Hz), 97.04, 166.49, 167.75, 169.14, 170.18,170.20. HRMS (M+Cs⁺) calcd C₁₇H₂₃O₁₁FCs 555.0279, found 555.0288.

Example 11 Larger Scale Synthesis of 18

Fluorosugar 17b (340 mg, 2.25 mmol), sodium pyruvate (2.074 g, 28.9mmol), dithiothreitol (1.7 mg), NaN₃ (2.3 mg), phosphate buffer (pH 7.5,50 mM, 1.12 mL) was added to the enzyme solution (3.0 mL, 24 U). Afterthe pH was adjusted to 7.5, the volume was made up to 10.0 mL. Themixture was stirred under N₂ at room temperature for 7 days. The pH waslowered to 2.5 by addition of Dowex 50W-X8 (H⁺ form) and the mixture waskept at 0° C. for 1 h. The precipitate was removed by centrifugation at23,000×g for 1 h at 4° C. Before the anion-exchange resin treatment, theexcess pyruvate was removed as follows. The mixture was diluted to 80 mLand the pH was adjusted to 6.5 by the addition of 2N aqueous ammoniasolution. The antifoam (Antifoam AF emulsion, Dow—Corning Nakaraitesque,10% emulsion in water, 0.32 mL) and pyruvate decarboxylase (Sigma P6810, 0.2 mL, 12.5 U) was added and the mixture was stirred at roomtemperature with bubbling of N₂ (1.5 L/min). The pH was monitored andoccasionally adjusted between 6.0 and 6.5, by addition of Dowex 50W-X8(H⁺ form). The decarboxylase was periodically added to the mixture (each0.2 mL) at an interval of 30 min, to avoid the denaturation which iscaused by the rapid formation of acetaldehyde. The total amount of theenzyme was 3.2 mL (200 U). The reaction mixture was further stirredovernight. Then the mixture was centrifuged, and the supernatant wasdiluted to 100 mL and applied to a column of Dowex 1-X8 (20-50 mesh,bicarbonate form, bed volume, 100 mL). The pH of the eluent and washingswas re-adjusted to 5.5 and further applied to the same column to ensurethe adsorption of desired product. After washing with water, the desiredproduct was eluted with a linear gradient from 0 to 0.3 M of ammoniumbicarbonate. The product was further purified by Biogel P-2 column (bedvolume 20 mL) to give 192 mg (33i) of 18. The ¹H NMR spectrum wasidentical with the sample mentioned above.

Example 12 Benzyl2,4,5,7,8-pneta-O-acetyl-3-deoxy-α-D-manno-2-octulosonate (20b)

A suspension of KDO ammonium salt monohydrate (160 mg, 0.59 mmol),acetic anhydride (3 mL), pyridine (3 mL), and4-(N,N-dimethylamino)pyridine (DMAP, 2 mg) was stirred overnight at roomtemperature. Ice-cooled water was added and the mixture was stirred for30 min. After dilution with water, the pH of the mixture was adjusted to3.5 by addition of Dowex 50W-X8 (H⁺ form). The resin was filtered off,and the filtrate was concentrated in vacuo. The residue was diluted witha mixture of chloroform and toluene and the solvent was evaporated. Thisprocedure was repeated three times to remove trace of water. The residuewas dissolved in anhydrous DMF. Benzyl bromide (161 mg, 0.94. mmol),Cs₂CO₃ (390 mg, 1.20 mmol), and tetrabutylammonium iodide (33 mg) wereadded and the mixture was stirred for 4 h at room temperature under N₂.The mixture was diluted with 0.5 N ice-cooled hydrochloric acid andextracted twice with a mixture of diethyl ether and toluene (1:1). Theorganic layer was successively washed with water, saturated aqueousNaHCO₃ and brine, dried over anhydrous Na₂SO₄ and concentrated in vacuo.The residue was chromatographed over silica gel (20 g). Elution withhexane-diethyl ether (2:1-1:1) afforded 15b, which was recrystallizedfrom diethyl ether to give 220 mg (70%) as colorless plates, mp 102-103°C. (lit.^(26b) mp 98-99° C.); [α]²⁶D+293° (c 1.0, CHCl₃) [lit.^(26b)[α]²⁵D+91.9° (c 0.9, CHCl₃). Its ¹H NMR spectrum (CDCl₃) was in goodaccordance with that reported previously by Nakamoto (Chem. Pharm. Bull.1987, 35, 4537). HRMS (M+Na⁺) calcd 561.1584, found 561.1602.

Example 13 2,4,5,7,8-Penta-O-acetyl-3-deoxy-α-D-manno-2-octulosonic acid(20a)

A mixture of 20b (220 mg, 0.41 mmol) and Pd—C (10%, 55 mg) in ethanol (3mL) was vigorously stirred under H₂ at room temperature for 1 h. Afterthe catalyst was filtered off, the filtrate was concentrated in vacuo.The residue was recrystallized from diethyl ether to give 20a (177 mg,97%) as fine needles, mp 132-133° C.; [α]²⁵D+374° (c 0.88, CHCl₃). Its¹H NMR spectrum (C₆D₆) was identical with that reported previously byUnger et al. (Carbohydr. Res. 1980, 80, 191).

Example 14 1,3,4,6,7-Penta-O-acetyl-2-deoxy-β-D-manno-heptose (21)

To a solution of acid chloride prepared from 20a (30 mg, 0.067 mmol) intoluene was added dropwise a solution of N-hydroxythiopyridone 22 (11mg, 0.09 mmol) and DMAP (2 mg) in toluene (0.5 mL) and pyridine (0.3 mL)at room temperature under N₂ in the dark. After stirring for 10 min,t-butylmercaptane (0.5 mL) was added and the mixture was irradiated withwhite light (tungsten lamp, 100 W) at room temperature. After stirringfor 10 min, N₂ was introduced to the mixture under a slightly reducedpressure to remove residual t-butylmercaptane for 30 min. Usual workupand purification by silica gel preparative TLC [developed withhexane-Et₂O (1:1)] afforded 21 (18.5 mg, 68%) as an oil, [α]²²D+36.8° (c1.85, CHCl₃); ¹H NMR (CDCl₃) δ2.000-2.150 (2H, m, H-2ax, H-2eq), 2.010(6H, s, acetyl), 2.082 (3H, s, acetyl), 2.119 (3H, s, acetyl), 2.137(3H, s, acetyl), 3.882 (1H, dd, J_(5,4)=1.5, J_(5,6)=10.0 Hz, H-5),4.115 (1H, dd, J_(7′,6)=4.5, J_(7′,7)=12.5 H, H-7′), 4.437 (1H, ddJ_(7,6)=2.5, J_(7,7′)=12.5 Hz, H-7), 5.073 (1H, ddd, J_(3,4)=3.0,J_(3,2eq)=5.0, J_(3,2ax)=12.5 Hz, H-3), 5.165 (1H, ddd, J_(6,7)=2.5,J_(6,7′)=4.5, J_(6,5)=10.0 Hz, H-6), 5.303 (1H ,dd, J_(4,5)=1.5,J_(4,3)=3.0 Hz, H-4), 5.748 (1H, dd, J_(1,2eq)=3.0, J_(1,2ax)=10.0 Hz,H-1); ¹³C NMR (CDCl₃) δ20.59, 20.59, 20.65, 20.65, 20.84, 30.35, 62.26,63.84, 67.32, 67.90, 71.62, 91.67, 168.60, 169.60, 169.83, 170.30,170.54. HRMS (M+Cs⁺) calcd C₁₇H₂₄O₁₁Cs 537.0373, found 537.0359.

Example 154-Acetamdo-1,3,6,7,8-Penta-O-acetyl-2,4-dideoxy-α-D-glycero-D-galacto-octose(24)

A 25 mL two-necked flask equipped with septum, micro-scale Dean-Starktrapp which was filled with molecular sieves 4A, and a reflux condenser,was used as the reaction vessel. A mixture of 23a (35.0 mg, 0.07 mmol),DMAP (12.3 mg, 1.5 eq), 22 (41.0 mg, 5.0 eq), triethylamine (19 μL) inCH₂Cl₂ (1 mL) was placed in the flask as above. To this was successivelyadded a solution of WSCI-Cl (20 mg) in CH₂Cl₂(1 mL) andt-butylmercaptane (0.5 mL). The mixture was stirred and irradiated withwhite light (tungsten lamp, 100 W) at room temperature for 5 h. Thereaction was worked up in a similar manner as described above. The crudeproduct was purified by silica gel preparative TLC [developed with ethylacetate-tetrahydrofuran (1:1)] to give 24 (8.7 mg, 27% from 23a) as anoil, [α]²²D+21.3° (c 2.87, CHCl₃); ¹H NMR (CDCl₃) δ1.908 (3H, s,N-acetyl), 1.915 (1H, ddd, J_(2ax,1)=10.3, J_(2ax,3)=11.5,J_(2ax,2eq)=12.4 Hz, H-2ax), 2.043 (3H, s, O-acetyl), 2.051 (3H, s,O-acetyl), 2.102 (3H, s, O-acetyl), 2.107 (3H, s, O-acetyl), 2.134 (3H,s, O-acetyl), 2.219 (1H, ddd, J_(2eq,1)=2.1, J_(2eq,3)=4.9,J_(2eq,2ax)=12.4 Hz, H-2eq), 3.764 (1H, dd, J_(5,6)=2.4, J_(5,4)=10.4Hz, H-5), 4.023 (1H, dd, J_(8,7)=5.5, J_(8,8′)=12.6 Hz, H-8), 4.062 (1H,ddd, J_(4,NH)=10.0, J_(4,3)=10.3, J_(4,5)=10.4 Hz, H-4), 4.389 (1H, ddd,J_(8′7)=2.6, J_(8′, 8)=12.6 Hz, H-8′), 5.127 (1H, ddd, J_(7,8′)=2.6,J_(7,8)=5.5, J_(7,6)=7.3 Hz, H-7), 5.058 (1H, ddd, J_(3,2eq)=4.9,J_(3,4)=10.3, J_(3,2ax)=11.5 Hz, H-3) 5.190 (1H, d, J_(NH,4)=10.0 Hz,NH), 5.391 (1H, dd, J_(6,7)=7.3, J_(6,5)=2.4 Hz, H-6), 5.646 (1H, dd,J_(1,2eq)=2.1, J_(1,2ax)=10.3 Hz, H-1); ¹³C NMR (CDCl₃) δ20.70, 20.70,20.75, 20.83, 20.83, 23.15, 35.09, 49.22, 61.98, 67.11, 70.23, 70.23,73.67, 91.19, 168.75, 169.90, 170.12, 170.36, 170.59, 170.88. HRMS(M+Cs⁺) calcd C₂₀H₂₉O₁₂NCs 608.0744, found 608.0750.

What is claimed is:
 1. A method of synthesizing a 7-8 carbon2-deoxy-aldose comprising the following steps: Step A: providing aprotected form of 8-9 carbon 2-keto-3-deoxy-onic acid with protectedside groups exclusive of the acid group; then Step B: condensing theacid group of the 8-9 carbon 2-keto-3-deoxy-onic acid of said Step Awith a thiohydroxamic acid to a form an O-acyl thiohydroxamateintermediate; then Step C: decarboxylating the O-acyl thiohydroxamateintermediate of said Step B by means of a radical mediateddecarboxylation for forming a protected form of the 7-8 carbon2-deoxy-aldose; and then Step D: deprotecting the protected form of the7-8 carbon 2-deoxy-aldose of said Step C for forming the 7-8 carbon2-deoxy-aldose.
 2. A method according to claim 1 wherein the 7-8 carbon2-deoxy-aldose is 2-deoxy-β-D-manno-heptose and wherein: in said Steps Aand B, the protected 8-9 carbon 2-keto-3-deoxy-onic acid is2,4,5,7,8-penta-O-acetyl-3-deoxy-α-D-manno-2-octoulosonic acid.
 3. Amehtod according to claim 2 wherein the 7-8 carbon 2-deoxy-aldose is4-amido-2,4-dideoxy-β-D-glycero-D-galacto-octose and wherein: in saidSteps A and B, the protected 8-9 carbon 2-keto-3-deoxy-onic acid is4-acetamido-1,3,6,7,8-penta-O-acetyl-2,4-dedeoxy-α-D-glycero-D-D-galacto-octose.4. A method of synthesizing a 7-8 carbon 2-deoxy-aldose according toclaim 1 wherein: in said Step B, the thiohydroxamic acid is2-mercaptopyridine N-oxide.
 5. A method of synthesizing a 7-8 carbon2-deoxy-aldose according to claim 4 wherein: in said Step C, the radicalmediated decarboxylation is effected by irradiation with light.